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CHARACTERIZATION OF Rltodotorula rubra TPl MUTANTS

by

Subhashini Mallidi, B.Sc.

Thesis submitted to the School of Graduate Studies

in partial fulfillment of the requirements

for the degree of Master of Science

Department of Biology

Memorial University of Newfoundland.

April, 2003

St. John's Newfoundland & Labrador Canada

ABSTRACT

Carotenoid pigments exist in nature and are widely distributed as colourants throughout the biological systems, such as microalgae, insects, birds, fish and crustaceans. They are responsible for interesting colours seen in various parts of these organisms, which play a great role in the biological functions like photoreception and photosynthesis. Carotenoids are mainly used as pigments for colouration of food products and pharmaceuticals. They also function as antioxidants and help in minimizing membrane-damage, and in controlling human diseases such as cancer, cataract and atherosclerosis.

Astaxanthin is a red orange carotenoid produced by aquatic organisms such as algae and is also found in yeasts like Phaffia rhodozyma and Rhodotorula rubra. It is used as a pigment in feed for salmon and shellfish and also enhances immune response of

fish and shrimp. Among yeasts, R. rubra TP l is a good source of red pigment and whole cells induce pigmentation in fish. It has been shown in earlier work that R.rubra bas faster growth rate, shorter incubation-time and yields more biomass than P.rhodozyma.

Further, previous feeding-trial experiments canied out using rainbow trout have been successful and therefore R. rubra TP 1 has economic potential.

In the current work the mutants Ml, M2 and M3 of R. rubra TPl were characterized and their properties compared with those of the wild type yeasts. The optimal pigment production was detem1incd by growing the mutants and wild type yeasts under different growth conditions, such as different substrates, temperatures, initial pH and light. The maximum pigment recovery was achieved by using different extraction methods which include French Press method, Freeze- dried cells, sonication and

enzymatic cell breakage. The spectrophotometer graph and Thin Layer Chromatograpy (TLC) techniques were used to estimate the total carotenoid concentration and to analyse the pigment in each sample.

The experimental results showed that light enhances pigment production. Yeast malt broth with peat extract as a nitrogenous source showed more biomass yield. Bacto czapex dox broth was found to be inhibitory to growth of the mutants of R. rubra TP 1.

The cells gave more pigment at 25 °C in the initial pH range of 5.0 to 7.0. The French press method was found to be more efficient to extract the optimum pigment for M 1, M2 and M3 with values 250.6, 254.4 and 193.2 J.tg/g, respectively. Mutant 2 alone gave higher recovery of the pigment with Freeze- dried method. Sonication method gave less pigment recovery. The enzymatic method with a pH of 7.0 for all mutants gave recovery values of 184.4, 164.2 and 129.4 J.tg/g for Ml, M2 and M3, respectively. The pigment analysis confirmed that all the mutants contain ,6-carotene, torulene and torularhodin carotenoids in their pigments.

TABLE OF CONTENTS

Abstract

Table of Contents List of Tables List of Figures List of Abbreviations Acknowledgements

CHAPTER!. INTRODUCTION 1.1 Manifestation of colour 1.2 Major pigment types 1.3 Functions of carotenoids 1.4 Applications of the catotenoids 1.5 Taxonomy of Rhodotorula rubra 1.6 Mutagenesis

1. 7 Astaxanthin

1.8 Significance of carotenoids

1.9 Carotenogic yeasts as sources of carotenoids 1.10 The genus Rhodotorula

1.11 Commercial Importance of Rhodotorula species 1.12 The Red yeast, Rhodotoru/a rubra TP 1

1.13 Description of R. rubra TPl

1.14 Potential commercial applications R. rubra TPl 1.15 Research objectives

CHAPTER 2. MATERIALS AND METHODS 2.1 Chemicals

2.2 Sources of microorganisms 2.3 Peat extract and cane molasses 2.4 Lysing enzymes

2.6.1 Preparation of media and inoculum

2.6.2 Growth cultures and harvesting of yeast cells 2.6.4 Growth on molasses and peat substrates 2.6.5 Growth measurement and generation times 2.6.6 Effect of temperatures on pigment production

2.6.7 Effect of initial pH ofthe growth media on biomass yield 2.6.8 Effect of light on growth on biomass yield

Page:

11 IV VI Vll

Vlll IX

4 5 6 8 8 10 12 14 15 16 17 17 18 19

20

21 21 21 21 22 22 22 23 23 24 24 25

2.6.9.1 Pigment extraction using French press 2.6.9.2 Pigment extraction, using Freeze-drying 2.6.9.3 Extraction by sonication method

2.6.9.4 Pigment extraction by using enzymes 2.6.9.4.1 Freeze and Thaw method

2.6.9.4.2 Effect ofThiol group on pigment recovery 2.6.1 0 Measurement of pigment procedure

2.6.11 Thin Layer Chromatography 2.6.12 Standard error in the mean

CHAPTER 3. RESULTS AND DISCUSSION 3.1.1 Growth on cane molasses and peat extract 3.1.2 Effects of the media on biomass yield 3 .1.3 Effects of temperature

3.1.4 Effects of initial pH 3.1.5 Effects ofLight

3.2 Effect of extraction methods on pigment recovery 3.2.1 French Press

3.2.2 Freeze-drying 3.2.3 Sonication

3.2.4 Extraction using enzymes

(i) Effect of lytic enzymes on pigment release (i i) Effect of Buffers

(iii) Effect of reducing reagents (iv) Effect of freezing and thawing

CONCLUSIONS REFERENCES

25 27 27 29 29 31 32 32 32

34 34 45

46 48

50 55 55 55 58 67 67 67

71 71

80

82

LlST OF TABLES

Page:

Table l. Biomass yield (giL) in wild type and mutant yeasts at various temperatures 39 Table 2. Rfvalues ofCarotenoids from various yeasts. 47 Table ^3. Effect of initial pH on biomass yields in yeasts. 50

Table 4. Absorption spectra of the yeast samples. 54

Table 5. Effects of lysing enzyme from different sources. on pigment recovery. 68 Table 6. Recovery of carotenoids by lytic enzyme (R. so/ani) in Tris-HCI buffer. 69 Table 7. Recovery of carotenoids by lytic enzyme (R. solani) in citrate phosphate

Buffer. 70

Table 8. Recovery of carotenoids by lytic enzyme (R. solani) in Dithiothreitol (DTT). 72 Table 9. Recovery of carotenoids by lytic enzyme (R sol ani) in Beta mercapto ethanol

(BME). 73

Table 10. Recovery of carotenoids by lytic enzyme (R. so/ani) by freeze and thaw

method. 74

Table 11. Recovery of carotenoids by enzymatic breakage and French press method. 79

LIST OP FIGURES

Page:

Fig. 1. Chemical structures of carotenoids 2

Fig. 2. Isoprenoid pathway 3

Fig. 3 Chemical structure of Polyene chain with a variation in the end groups 7 Fig. 4. French press procedure for pigment extraction 26

Fig. 5. Freeze drying procedure for extraction 28

Fig. 6. Enzymatic cell breakage procedure for pigment extraction 30

Fig. 7. Generation times of yeasts 35

Fig. 8. Growth of yeasts on YM-broth at 15 °C 36

Fig. 9. Growth of different yeasts on YM-broth at 25 °C 37 Fig. 10. Time needed for achieving stationary phase for yeasts in liquid cultures 38 Fig. 11. Effect of I %peat in YM-broth on biomass yield 40 Fig 12. Effect of2% peat extract in YM-broth on biomass yield 42 Fig 13. Effect of 3 % peat extract on biomass yield 4 3 Fig. 14. Effect of carbon source on biomass yield 44

Fig. 15. Effect of light on pigment production 51

Fig. 16. Effect of darkness on pigment production 52 Fig. 17. Effect of French press on pigment recovery in isolates 56 Fig. 18. Effect of Freeze-drying on pigment recovery in all the isolates 57 Fig. 19. Effect of sonication method on pigment recovery in all the isolates 59 Fig. 20. Effects of extraction methods on pigment recovery from TPl 60 Fig. 21. Pigment recovery from Mutant 1 under different extraction methods 61 Fig. 22. Pigment recovery from Mutant 2 under different extraction methods 62 Fig. 23. Pigment recovery from Mutant 3 under different extraction methods 64 Fig. 24 Pigment recovery from Rm yeast, under various extraction methods 65 Fig. 25. Pigment recovery from Rt yeast under various extraction methods 66 Fig. 26. Plot of substrate concentration versus optical density for isolates 76 Fig. 27. Plot of enzyme concentration versus optical density for isolates 77

Yeast Malt Broth

Bacto Czapex Dox Broth Gram,/Litre

Micro gram/ gram

Thin Layer Chromatography Nanometer

Beta mercapto ethanol Dithiothreiotol

Degree centigrade

ABBREVIATIONS

YMB

BCDB giL JLg/g TLC nm BME

oc

ACKNOWLEDGEMENTS

I express my sincere gratitude to my supervisor Dr. T. R. Patel for the supervision of the research project and for his advice, encouragement and financial support. I also thank the other members of my supervisory committee, Drs. A.M. Martin and Jyoti Patel for their interest in my research project and for making useful suggestions.

I am grateful to the School of Graduate studies, Memorial University of Newfoundland, for their financial support.

I acknowledge the technical assistance provided by Peter Earle, Garry Colins, Kevin Snow, Kathy Anthle and William Brown of the Biology Department. The administrative cooperation provided by Shena Quinton, Patricia Squires, Shirley Kenny and Christine Everson is also appreciated.

Special thanks are extended to my past and present colleagues in Dr. Patel's Laboratory and several of my friends who in diverse ways made my stay in St. John's enjoyable.

I appreciate and acknowledge the affection, encouragement and inspiration provided by my loving parents, aunt, uncle and other members of my extended family.

CHAPTER-1

INTRODUCTION

The quality of food, aside from the microbiological aspects, is generally based on its colour, flavour, texture, and nutritive value. Depending on the particular food, these factors may be weighted differently in assessing overall quality. However, one of the most important sensory quality attributes of food is colour, because no matter how nutritious, flavourful, or well textured a food is, it is unlikely to be accepted unless it has the appropriate colour. The acceptability of food is reinforced by economic worth since in many cases raw food materials are judged on the basis of their colour.

Pigments are chemical compounds which reflect only certain wavelengths of visible light, making them appear "colourful". Flowers, corals, and even animal skin contain pigments, which give them their colors. The ability of pigments to absorb light of certain wavelengths is more important than reflection by them.

The term "pigment" is used to refer to a material of known or unknown physical state or to an unanalyzed coloured material (Sangha, 1994). Colours of various carotenoids are related to the number of alternating carbon-carbon double-bond pairs in the long polyene chain of the molecule, known as the chromophore (Fig. 1).

Specifically, light energy is absorbed by the carotenoid polyene system between 400 - 700 nm, and is converted into vibrational energy and heat. Each carotenoid has a unique resonance in this regard (Fox, 1976) through the isoprenoid pathway (Fig. 2) and they produce diverse compounds such as essential fatty acids, steroids, sterols, and vitamins A, D, E, and K.

A.STAXANTtnN

hOpRMwUI

{a)

(b) '

CAROTENE. 8-

CAROTENE. aamma·

(c) (d)

TORUL&NE TORULAIUIOOJN

"'

(d) (e)

Fig. I Chemical structure ofCarotenoids (Hari eta/., 1992)

C2 1-Deoxy-D-xylulose-5-P

1

C5 lsopentyl pyrophosphate

C 10 Geanyl pyrophosphate

1 1

C15 Famesyl pyrophosphate

1

C20 Geranylgeranyl pyrophosphate

1

C2 1-Deoxy-D-xylulose-5-P

1

C 40 Phytoene

1

C40 carotenes hydroxylase

Phytol

1

Chlorophy 11

C40 Xanthophylls (lutein) (seaxanthin)

Within the various classes of natural pigments, the carotenoids are the most wide spread and structurally diverse pigmenting agents. They are responsible, in combination with proteins, for many of the brilliant yellow to red colors in plants and the wide range of blue, green, purple, brown and reddish colors of fish and crustaceans. The general distribution and metabolic pathways of carotenoids have been extensively detailed (Goodwin 1984). Carotenoids are widespread throughout biological systems. They are found in the plants, algae, bacteria, animals and fungi (Goodwin, 1980). Several species of yeasts produce carotenoids and are grouped as the 'red yeasts'. These carotenogenic ascomycetes, basidiomycetes and deuteromycetes all tend to accumulate predominantly hydrocarbon carotenoids, such as beta-carotene and gamma-carotene (Goodwin, 1980).

1.1 Manifestation of colour:

Colour is displayed by organisms m two ways, namely, (1) physically, by colourless particles or ultramicroscopic structures called "schemochromes", and (2) chemically, by naturally occurring chemical substances possessing a coloured molecule, called "biochromes" (Fox, 1979).

Schemochromes are exhibited by both colourless, randomly scattered, light- diffracting submicroscopic bodies. These give rise to the Tyndall blues of scattering and various striations or ultrathin successive films or layers which resolve incident light into its components producing interference colors (Fox, 1979).

Biochromes absorb wavelength, while reflecting and/or transmitting other wavelengths of visible light (Fox, 1979). The structural feature of a biochrome

responsible for the absorption of light is the chromophore. For example, in carotenoids the chromophore is the conjugated carbon-carbon double bond system.

Other functional groups or substituents in a biochrome, which possess the ability to modify the absorption maximum of the molecule are termed auxochromes. Vision in humans and animals is a complex chemical phenomenon. The human eye, for example is roughly spherical with an opening to admit light, which falls on a rear surface lined with millions of cells. The molecules responsible for vision are attached to the cells. Discrimination between colours is possible because cone cells occur in three groups:

those receptive to blue light, those receptive to green light and those receptive to yello- red light. Each type can absorb light in a range around its primary color. When an object absorbs these wavelengths (visible range 400 and 750 nm), certain molecules within the object become excited. A molecule is excited when one of its outer orbital electrons is raised to a higher orbital. These electron transitions are characteristic of most biological materials but are particularly pronounced in biochromes (Needham, 1974a).

1.2 Major Pigment Types:

There are six major groups of pigments occurring in biologial systems. These are carotenoids , tctrapyroles, indolic biochromes, N-heterocyclic biochromes (other than tetrapyrroles), oxygenous heterocyclic biochromes (the flavonoids) and quinones.

Carotenoids are nature's most widespread pigments, with the earth's annual biomass production estimated at 100 million tons (Fennema, 1996). In nature over 560 carotenoid structures have been identified and compiled. They derive their names from

the fact that they constitute the major pigment in the carrot root, Daucus carota, one of the first foods observed to possess this class of pigments (Kiaui eta/., 1981 ).

1.2.1 Functions:

Most of the functions of carotenoids are a consequence of their ability to absorb visible light. It has been established that carotenoids play a role in photoreception (vision), photosynthesis, photoprotection, phototaxis and integumental colors (Burnett, 1965; Needham, 1974; Goodwin, 1980; Sangha, 1994; Britton et a/., 1995). The luminous carotenoid colours of tropical fish are not only keys for species identification and mating signals they have significant physiological roles as well. The seasonal astaxanthin levels in the carapace have shown that the eggs parallel with the exposure to sunlight, indicating that the carotenoids serve to protect external proteins and eggs from ultraviolet exposure. Beta-carotene is converted to vitamin A, which is required for the biochemical processes involved in vision (Goodwin, 1980). Furthermore, vitamin A plays an important role in the growth, development, and integrity of mucous surfaces.

However, the majority of research concerning astaxanthin and other carotenoids has been aimed at its role in photoprotection and as an antioxidant in quenching of oxygen radicals.

Carotenoids owe their color to the absorption of light by the feature of their molecular structure known as the 'chromophore'. In most carotenoids the chromophorc consists entirely of a conjugated system of carbon-carbon double bonds, referred to as the 'polyencchain' (Fig. 3). It is possible to have up to 15 conjugated double bonds in the chromophore of a C40 carotenoid, although structures with 7 to 11 such bonds are

~ R

R ~

~ 0::' ^C(

0( C)( ⁰⁽

OH

«

HOi::( 1::(

0 0

.ct~ p& HOi:X§

HO ~

Fig. 3. Chemjcal structure of polyene chain with a variation in the end groups.

(Weedon et al., 1995)

more common. Other features of carotenoid molecules that may constitute part of the chromophore are triple bonds, terminal allene groups, substituted phenyl end groups, and carbon-oxygen double bonds (Weedon et al., 1995).

1.3 Applications of Carotenoids:

Carotenoids have commercial application in various industries such as aquaculture, food industry, pharmaceutical, cosmetic, and medicine (Bauernfeind and K.laui, 1981; Munzel, 1981; Sangha, 1994 ). The use of carotenoids as pigments in aquaculture is well documented. It appears that their broader functions include a role as an antioxidant and provitamin A activity as well as enhancing immune response, reproduction, growth, maturation and photoprotection. An extensive body of data stresses the vital role of carotenoids in the physiology and overall health. It concludes that carotenoids are ·essential nutrients that should be included in all aquatic diets at a minimum level of5 -10 ppm (Torrissen, 1989).

1.4 Taxonomy of Rhodotorula rubra:

Yeast is defined as a unicellular fungus that reproduces by budding or fission (Kreger van-Rij, 1984). Yeasts are taxonomically diverse and classified in the division Eurnycota, which includes the classes Ascomycotina, Basidiomycotina and Deuteromycotina (Kreger van-Rij, 1984). The ascomycetes are recognized as unpigmented yeasts possessing asci with ascospores, and reproduce by holoblastic budding (Kratochvilova, 1990).

The system of taxonomy used today is the result of the development and integration of various avenues of approach to the problem of yeast identification and classification (Lodder, 1970). Morphological and reproductive attributes are utilized to decide the main taxonomy- to designate higher taxa while physiological evidence is used to differentiate lower taxa, and in particular, species classification. Most of these characteristics are defined based on a particular test, such as fermentation and assimilation (Kreger-van-Rij, 1984). The isolation of the mutants of Rhodotourla rubra TP 1 used in this work has been reported (Acheampong, 2000).

The carotenoid-producing yeasts include genera such as Cryptocccus, Rhodotorula, Rhodosporidium, Sporidiobo/us, Sporobolomyces, Phaffia (Johnson and

Lewis, 1979) and Saitoella (Komagata et al., 1987). Yeasts belonging to the genera Cryptococcus, Rhodotorula, Rhodosporidium, Sporidiobolus, and Sporobolomyces

typically contain {J-carotene, ')'-carotene, torulene and torularhodin as major carotenoids (Simpson et al., 1971). The genera Rhodospridium and Rhodotorula may also produce carotene, phytoene, and phytofluene, 2-hydroxyplectaniaxanthin have been found in a strain of Rhodotorula aurantiaca (Lui et al., 1973). Some species of Rhodotorula also synthesize I)-carotene, IJ-zeacarotene and plectaniaxanthin, which are also found in Cryptococcus laurentii (Lui eta!., 1973).

The yeast Phaffia produces astaxanthin as its most abundant carotenoid. Other characterized carotenoids are I)-carotene, -y-carotene, neurosporene, lycopene, echinenone, 3-hydroxyechinenone, 3-hydroxy-3', 4'-didehydro-1)-carotene- 4- 1 and phoenicoxanthin (Andrewes et al., 1976).

1.6 Mutagenesis:

Several methods are available for genetic manipulations of biological cells.

Newer techniques include protoplast fusion, pulsed field electrophoresis and recombinant DNA techniques. However, difficulty arises in applying these methods when genetic information of a species is lacking. More fundamental approaches for strain improvement involve genetic mutations (Crueger and Crueger, 1989).

To enhance the potential of a microorganism, the genotype can be manipulated by inducing mutations in the genome. Common mutagenic agents include ultraviolet and ionizing radiations and chemical agents. These affect non-replicating DNA and cause frame-shifts in DNA and base substitution by analogs (Crueger and Crueger, 1989).

Short wavelength ultraviolet rays between 200 - 300 nm, with an optimum wavelength at 265nm, are effective in causing mutations. The absorption maximum of DNA is 265 nm. The most important products of this type of radiation are pyrimidine dimers, formed between adjacent pyrimidine bases on complementary strands of DNA.

Long wavelength ultraviolet rays between 300 - 400 nm are less lethal mutagens.

However, if cells are exposed to this type of radiation in the presence of various dyes, increased mutation frequency is induced (Crueger and Crueger, 1989). Ionizing radiation includes x-rays, y-rays, P-rays. These types of radiations are seldom used for mutagenesis as the rays cause a much greater percentage of single and double strand breaks in DNA than the other mutagens, which can result in major structural changes in the chromosome. A variety of chemicals are known mutagens and are used in genetic studies. These chemicals are classified according to their mode of action. Frame-shift mutagens intercalate into the DNA molecule, causing errors in the reading frame and

result in the formation of faulty proteins or no proteins at all. Examples of this type of mutagen are acridine dyes, such as acridine orange, proflavine and acriflavine. Although useful in research, frame-shift mutagens are not very suitable for isolation of mutants in strain development, because they have little or no mutagenic effect in bacteria and yeasts

(Crueger and Crueger, 1989).

Base analogs, such as 5-bromouracil and 2-aminopurine, act as mutagens by being incorporated into replicating DNA in place of the corresponding bases thymine and adenine because of their structural similarity. These cause transitions to occur, resulting in the wrong base pair being incorporated into the replicated DNA. Conditions for the development of this type of mutants are costly and as such, base analog mutagens are rarely used in practical applications.

Many carotenogenic, or red yeasts have also been genetically altered using N- rnethyi-N-nitro-N-nitrosoguanadine (NTG). An et al., (1989) evaluated the effectiveness of UV light, etbylmethanesulfonate (EMS) and NTG in generating greater pigment producing mutants of Pha.ffia rhodozyma. NTG was reported to be the best mutagen.

However, most of the mutants were unstable. In another attempt to obtain hyper pigment producing mutants, Lewis eta/. (1990) exposed Pha.ffia rhodozyma to NTG and then screened the astaxanthin-overproducers using beta-ionone. Acheampong (2000) successfully treated Rhodotoru/a rubraTP 1 with NTG in order to produce mutants with enhanced pigmentation and a better capacity to utilize cheaper substrates for growth.

1.7 Astaxanthin:

Astaxanthin is the main carotenoid pigment found in some aquatic animals. This red-orange pigment is closely related to other well-known carotenoids such as beta- carotene or lutein, but has a stronger antioxidant activity (10 times higher than beta- carotene). Studies suggest that astaxanthin can be 1000 times more effective as antioxidant than vitamin E. In many of the aquatic animals where it can be found, astaxanthin has a number of essential biological functions, ranging from protection against oxidation of essential polyunsaturated fatty acids to enhance immunity and growth. In species such as salmon or shrimp, astaxanthin is even considered as essential for normal growth and survival, and has been attributed to have vitamin-like properties.

Some of these unique properties have also been found to be effective in mammals and open very promising possibilities for nutritional and pharmaceutical applications of astaxanthin in humans. It can be found in many of seafoods such as salmon, trout, shrimp, lobster and fish eggs. It is also found in a number of bird species. Astaxanthin cannot be synthesized by animals and must be provided in the diet as is the case with other carotenoids. While fish such as salmon are unable to convert other dietary carotenoids into astaxanthin, some species such as shrimp have a limited capacity to convert closely related dietary carotenoids into astaxanthin, although they will benefit strongly from being fed astaxanthln directly. Mammals are also unable to synthesize astaxanthin. Some microorganisms can be quite rich in astaxanthin.

A ubiquitous micro-algae, Haematococcus pluvialis is believed to be the organism, which can accumulate the highest levels of astaxanthin in biological system.

The function of astaxanthin appears to be to protect the algae from adverse environment

changes, such as increased UV -light photoxidation and evaporation of the water pools in which it lives. Haematococcus algae can accumulate as high as 10 to 30 g of astaxanthin per kg of dry biomass. This level is 100 to 3000 fold higher than in salmon fillets. Some strains have even been observed to accumulate as much as 70 to 80 g of astaxanthin per kg of dry biomass. Esterified astaxanthin from Haematococcus pluvialis alagal meal is the preferred form in several oral prophylactic and therapeutic formulations for muscular dysfunction, such as exertional rhabdomyolysis (also known as exertional myopathy, tying-up syndrome, azoturia, or Monday morning sickness) in horses (Lignell, 1999), as well as for mastitis (mammary inflammation) in dairy cows (Lignell, 1999).

Astaxanthin is one of a group of natural pigments known as carotenoids. The astaxanthin molecule is similar to that of the familiar carotenoid, beta-carotene. The small differences in structure of these confer large differences in the chemical and biological properties of these two molecules. In particular, astaxanthin exhibits superior antioxidant properties to beta-carotene in a number of in vitro studies (Krinsky, 1992).

Higher survival rate in red sea-bream was found to be that astaxanthin enhanced liver cell structure. Glycogen storage in red tilapia increases fertilization and survival rates of eggs. Higher growth rates during the early-feeding period of young salmonids have all been associated with dietary astaxanthin supplementation (Sommer et a!. 1991; Torrissen and Christiansen 1995; Kawakami eta!. 1998). When astaxanthin was included in poultry feeds, dietary astaxanthin was reported to improve egg production, the general health of hens and also increase in the hatching percentage, resistance to Salmonella infection, and shelf life of eggs. (Lignell et al. 1998).

1.8 Significance of Carotenoids:

The food and pharmacological industries are potential users of large amounts of natural antioxidants. One of the advantages for the food industry is that these antioxidants may be used as preservatives against both enzymatic and spontaneous oxidation of foods, thereby extending their shelf life. Astaxanthin, which belongs to the carotenoid group, is a very valuable natural red dye used as a feed additive for deepening the pigmentation of salmon and organic chicken eggs. Initial results also show that astaxanthin is a promising cancer preventing agent and hence has potential for use as an additive for promoting good health (Tanaka, 1995).

In nature, like other pigments, astaxanthin is synthesized only by microalgae and then passed up the food chain. Salmon and other marine animals cannot make the compound themselves and must get it in their food. Traditionally astaxanthin has been added to commercial aquaculture diets to improve the pigmentation of the flesh of fish.

Thjs use remains by far the largest market in terms of volume and market value.

However a number of studies (Klaui H. and Bauernfeind, J.C., 1981) have shown that astaxanthin was muc_h more than a pigment and in fact had vitamin-like properties. As a result, astaxanthin is now also used to enhance the immune response of fish and shrimp to secure maximum survival and growth. Recent studies (Ito et a!., 1986) with young shrimp and other fish species have shown a superior uptake of natural astaxanthin from microalagae compared to the synthetic form. Another reason for aquaculturists to prefer natural astaxanthin is the growing demand from consumers for fish being fed natural pigments, identical to those fish that acquire natural astaxanthin from the environment.

1.9 Carotenogic Yeasts as sources of Carotenoids:

The pink to red color of the flesh of salmon ids is an important factor in consumer preference for coloured fish. Colour is not an intrinsic component of the fish, but results from the deposition of dietary carotenoids. Astaxanthin is an abundant carotenoid in the marine environment. Salmonids, like most animals, are unable to synthesize or biologically transform carotenoid precursors into the pigments found in their tissues.

Wild salmon obtain their carotenoids from marine zooplankton, nekton, and their natural foods. Pen-raised salmonids, in tum, must derive this pigmentation from sources in their feed.

The dominant pigment source in aquaculture is synthetic astaxanthin and canthaxanthin, commercially produced by Hoffman La Roche (Basle, Switzerland), which are marketed under the trade names of 'Carophyll pink' and 'Carophyll red', respectively (Torrissen et al., 1989). However, the use of synthetic feed colorants is quickly declining due to strict regulations and the increasing reluctance of consumers to accept chemicals as food additives.

In recent years, yeasts have been used as a pigment source for fish. The species Phaffia rhodozyma possesses high levels of carotenoids, of which astaxanthin is the most abundant. In feeding trials, the incorporation of this yeast's pigment into the diets has achieved high levels of pigment deposition in rainbow trout, lobsters and salmon (Johnson and Lewis, 1977). However, three major obstacles have prevented the commercial use of Phaffia rhodozyma as a natural source of carotenoids in fish feeds: a rigid cell wall, which limits the pigment extractability, a slow growth rate and poor digestibility of the whole Phaffia cells by the fish.

A strain of the Rhodotorula species, Rhodotorula rubra TPl, was also found to be a good source of pigments for rainbow trout. Unlike Phaffia rhodozyma, whole cells of Rhodotorula rubra TPl were able to induce pigmentation. In addition, this has been found to have a faster growth rate and easier pigment extractability than Phaffia rhodozyma (Sangha, 1994).

1.10 The Genus Rhodotorula:

The genus Rhodotorula belongs to the class Deuteromycotina, family Cryptococcaceae (Kreger van-Rij, 1984) and sub-family Rhodotoruloideae (Ladder and Kreger-van Rij, 1954).

Yeasts are classified in the family Cryptococcaceae by the constant presence of budding cells- although a pseudomycclium, true mycelium and arthrospores may be formed. Culture cells are hyaline, red, orange or yellow due to carotenoid pigments, and are seldom brown or black. Dissimilation is strictly oxidative or oxidative and fermentative (Kreger van-Rij, 1984).

Members of the genus Rhodotorula have ovoidal, spheroidal or elongate cells.

They reproduce vegetatively by multilateral budding and variants of some species form psuedohyphae or true hyphae. Neither ascospores nor ballistospores are formed. Red or yellow carotenoid pigments are synthesized in malt agar cultures (Kreger van-Rij, 1984).

Regarding culture appearance, some strains appear mucoid due to capsule formation, while others seem pasty or dry and wrinkled (Kreger-van Rij, 1984 ).

1.11 Commercial Importance of Rhodotorula Species:

The metabolic capabilities of some Rhodotorula species have indicated possible applications of this genus in the commercial industry. Two Rhodotorula rubra strains were found to degrade 4-hydroxy-benzoate and as such could be used in oil sludge treatment (Wright and Ratledge, 1991 ).

Ogrydziak (1993) reported the production of extra cellular proteases by a strain of Rhodotorula rubra. It was proposed that these proteases could be used to degrade the proteins responsible for protein hazes that form in wines and beers during storage.

1.12 Tbe red yeast, Rhodotorula rubra:

The species Rhodotorula rubra was first disvovered in 1889 by Demme under the name Saccharomyces ruber. Like all Rhod.otorula species, ascospores or ballistospores are not produced and reproduction is by multilateral budding. As described by Kreger van Rij (1984), Rhodotorula rubra assimilates glucose, sucrose, trehalose, raffinose, D- xylose, ribitol, melezitose and succinic acid. Galactose, maltose, cellobiose, L-arabinose, D-ribose, L-rhamnose, D-mannitol and citric acid are assimilated by some strains while lactose, soluble starch, erythritol, inositol, melibiose and nitrate are not assimilated (Kreger van Rij, 1984).

Cells grown in malt extract or on malt agar vary from short ovoidal to elongate, 2- 5.5 nm in width, and occur singly, in pairs, short chains or in clusters. Colony color ranges from deep coral to pink or salmon-colored. Colony surface is glistening and usually smooth, but is sometimes reticulate, corrugated and the texture varies from soft to mucous Kreger van Rij, (1984).

The carbohydrate patterns of Rhotorula rubra whole cell hydrolysates show the presence of fucose and mannose as the dominant sugars in this yeast, while hexitol and pentitol also occur in high concentrations (Weijman and Miranda, 1988). The total lipid content of Rhodotorula rubra is about 6.0% of dry weight, with palmitic acid, oleic acid and linoleic acid as the major lipids (Perrier et al., 1995). This carotenogenic yeast also contains about 100 mg carotenoids/g dry weight, which includes beta-carotene, beta- zeacarotene, torulene and torularhodin as the major components (Perrier et al., 1995).

The G+C content is 60-63.5 mol% (Nakase and Komagata, 1971).

1.13 Description of Rhodotorula rubra TPl :

Rhodotorula rubra strains have been isolated from leaves, flowers, soil, atmosphere and marine sources (Cook, 1958; Ingram, 1955; Kreger van Rij, 1984).

Recently a new strain has been isolated from yogurt (Hari et a!., 1992). A new strain of red yeast contaminating a home-fermented yogurt was isolated and, using the Analytical Profile Index (API) clinical yeast system, was identified as Rhodotorula rubra

(Hari et a!., 1992). The results confirmed by Microcheck Inc. Northfield, VT using a technique involving cell wall fatty acid analysis. The isolate was named Rhodotorula rubra TP 1 (Hari et al., 1992). Rhodotorula rubra mutants were isolated by Achempong (2000). He used three different mutagens including UV irradiation, ethyl methane sulfonate (EMS) and nitrosoguanadine NTG. He found NTG to be a better mutagen and he was able to isolate 8 mutants of R.rubra TPl.

Like other Rhodotorual rubra strains, Rhodotorula rubra TP 1 does not form ascospores or ballistospores, and reproduces by multilateral budding (Sangha, 1994).

However, in one study, Sangha, (1994) observed the presence of ascospores in this strain of yeast. As such, this experiment needs to be repeated. As described by Hari et a/., (1992). Rhodotorula rubra TPl assimilates melezitose, melebiose, maltose, mannitol, trehalose, D-ribitol, raffinose, citric acid, sucrose, arabinose, 0-xylose, succinic acid, soluble starch, galactose, and nitrate. It is unable to assimilate glucose, erythritol, inositol, rhamnose, cellobiose, and lactose.

Cells grown in yeast extract/malt extract (YM) broth are circular or ellipsoidal and average 2 to 4 nm in diameter. Colony color is best described as salmon-colored and the colony surface is glistening and smooth. The absorption spectrum of the pigment from Rhodotorula rubra TPl shows that the pigment belongs to the family of carotenoids. Rr values of the pigment on a thin-layer chromatography plates were similar to those obtained for standard astaxanthin, while a mass spectrometry analysis showed a molecular mass similar to that of astaxanthin (Hari eta/. 1992).

1.14 Potential Commercial Applications of Rhodotorula rubra TPl:

Sangha (1994) found Rhodotorula rubra TPl to be an efficient source of pigments and nutrients for aquacultured rainbow trout. The yeast was found to be more economically favorably over Pha.ffia rhodozyma, which has also been successful in pigmenting pen-raised salmonids. However, Rhodotorula rubra TP I has a faster growth rate with greater levels of pigment production compared to Phaffia rhodozyma under similar conditions of growth. Moreover, whole cells of R. rubra TP 1 were able to pigment rainbow trouts but cells of Ph. rhodozyma showed no pigmentation (Hari et al.

1993). They also found that Rhodotorula rubra TP I could be successfully grown on

various industrial and agricultural by-products for biomass production. This has important implications, as the cost of growing sufficient amounts of yeast cultures for commercial use has always been a concern. These raw material by-products are readily available, relatively low in cost while pure sugars like glucose and sucrose, which are often used for microbial growth in laboratory situations, are too expensive for use on an industrial scale.

1.15 Research Objectives:

An increased production of carotenoids by microorganisms such as red yeasts will make its industrial applications cost effective and competitive. With this in mind, mutant strains of R. rubra TP I were examined with the following objectives:

(i) To determine optimal growth conditions for pigment production under the influence of pH, light, temperatures, and different sources of carbon and nitrogen.

(ii) To investigate efficient methods for optimal extraction of pigments from mutants and parent cells using Freeze-drying, French Press, Sonication and enzymatic cell breakage methods in order to determine their efficiency on pigment recovery.

CHAPTER2

METHODS AND MATERIALS 2.0 Materials

2.1 Chemicals:

Acetone, dimethyl sulphoxide (DMSO), sodium chloride, sulphuric acid, sodium hydroxide, petroleum ether, hexane, iodine crystals, citrate phosphate, ethyl methane disulphate (EDTA), trizma base (Tris HCI), dithiothreitol and beta mercapto ethanol which were purchased from Fisher Scientific Company Ltd., Fair Lawn, N.J., U.S.A. All the chemicals were of Analar Grade and were used without further purification.

2.2 Sources of Microorganisms:

The test strain used in the experiments was Rhodotorula rubra TPI from earlier collection from Dr. T. R. Patel's Laboratory, Department of Biology, Memorial University of Newfoundland (MUN), NL, Canada. Mutants strains, Mutant 1 (MI), Mutant 2 (M2) and Mutant 3 (M3) were isolated earlier by Acheampong (2000) working in the same laboratory. These mutants, maintained on Rose Bengal Agar plates (purchased from Difco Laboratories, Detroit, MI, U.S.A.) and stored at 40" C. These were transferred once a month onto new plates. Rhodosporidium toruloids ( 1 0657) and Rhodotorula minuta ( 1 0658) were from American Type of Culture Collection (ATCC).

2.3 Peat Extract and Cane Molasses:

Peat extract was a gift from Dr. A.M. Martin's Laboratory, Department of Biochemistry, MUN. Cane molasses was procured from Lalle Nand Inc., Montreal, PQ.

2.4 Lysing Enzymes:

Lysing enzymes from Trichoderma harzianum, Aspergillus species, Cytophaga species and Rhizoctonia so/ani were purchased from Sigma Chemical Company, St.

Louis, MO, U.S.A.

2.5 Media:

Yeast Malt Broth (YMB), Potato Dextrose Agar (PDA), Bacto Czapex Dox Broth (BCDB) and Rose Bengal Agar base (RBA) were purchased from Difco Laboratories.

2.6 Methods:

2.6.1 Preparation of Media and Inoculum:

Yeast malt (YM) broth was prepared according to the instructions given by the manufacturer. Loop-fulls of yeast from RB agar plates were aseptically added to 10 ml of saline water and vortexed. This suspension was used for inoculating growth media.

2.6.2 Growth of Cultures and Harvesting of Yeast Cells:

Yeast cells were grown in liquid media of different types. Erlenmeyer flasks (2 L) containing 500 ml liquid YM broth were inoculated with yeast suspension and were incubated at 28° C for 5 days in a Psychrotherm Temperature Control Shaker (New Brunswick Scientific Co. Inc., Edison, New Jersey, U.S.A). The cultures were agitated at 150 rpm. Liquid cultures were centrifuged at I 0,000 rpm for 10 minutes to pellet the cells. The pelleted cells were used for pigment extraction after washing three times in a saline solution.

2.6.3 Optimization of Growth Conditions:

Different growth conditions such as substrate concentration, initial pH of the culture medium, temperature, light, fermentation time, initial optical density of the inoculum and agitatio~ speed were tested to detennine the optimum growth parameters for the mutants of R.rubra TPl.

2.6.4 Growth on Molasses and Peat Substrates:

(i) Crude molasses diluted at a ratio of I: 10 was used to determine the effect of carbon source on pigment production. YM broth medium (500 ml) contained 50 ml cane

molasses, as a supplement of carbon source was incubated in 2 L flasks at 28° C on a shaker at a speed of 150 rpm for 4 days. Aliquots (1 ml) were removed in 3 hour intervals and optical density was measured on a Pharmacia LKB Novaspec II spectrophotometer at a wavelength of 600 nm.

(ii) Peat extract diluted at a ratio of I : 10 was used to detennine the effect of nitrogen source on pigment production. YM broth (500 ml) contained 50 ml peat extract as a supplement of nitrogeneous source. Broth cultures were incubated at 28° C on a shaker at a speed of 150 rpm. Aliquots (1 ml) were removed in 3 hour intervals and the optical density was measured using a spectrophotometer.

2.6.5 Growth Measurement and Generation Times:

Growth and biomass of the wild type R.rubra TPI and the mutants were measured using methods such as (i) optical density measurement, (ii) dry weight and wet weight determinations. Growth in liquid media were examined as follows: YM broth (500 ml) in

2 L flasks were incubated at 28° C on a shaker (at 150 rpm) after inoculation with 2 ml suspension of the yeast. Aliquots (1 ml) were removed at 3 hour intervals and optical density readings were used to establish growth curves. Generation times were calculated using the logarithmic growth phase of each culture. Readings were taken in triplicates for each yeast sample. After 5 days of incubation the yeast cells were collected by centrifugation in a pre-weighed centrifuge bottles, washed twice with saline, weighed and dried in a hot air oven at 80° C (Oven, Blue M electric company, Blue Island, Illinois, U.S.A.). The dry weight was recorded after three constant readings were observed. Growth table gives the generation time values given by T = (t2-t1) I log (y/x), where x = cells/ml at time t1 andy= cells/ml at time t2.

2.6.6 Effect of Temperature on Pigment Production of the R. rubra TP 1 mutants:

To study tbe effect of different temperatures on growth and pigment production, the culture flasks (70 ml Hquid medium in 250 ml flasks) were incubated at 15, 20, 25, 28, 30 and 35° C on a shaker for 5 days. Growth was determined by wet weight and dry weight of the cells.

2.6. 7 Effect of Initial pH of the Growth Media on Pigment Production of Mutants:

To study the effect of different initial pH on growth and pigment production, the pH of the growth medium was adjusted between pH 3 to 10. This was achieved by adding NaOH (l M) or by adding HCI (1 M) to the broth. Yeast cell suspension (2 ml) was added to 100 rnl of YM broth contained in 250 ml flasks. These flasks were

incubated at 28° C on a shaker for 5 days. Growth was detennined by the optical density method as well as by the wet weight and dry weight methods.

2.6.8 Effect of Light on Growth and Pigment Production of R.rubra TPI Mutants:

To study the effect of light on growth and pigment production, the culture flasks were incubated in dark or in the presence of light on a shaker at 28° C for 5 days.

Biomass-yield was obtained by using wet weight and dry weight methods.

2.6.9 Pigment Extraction:

2.6.9.1 Extraction using French Press:

Wet cells ( 4 g) were placed in the French Press Cell (SLM Instruments, Chicago, Illinois, U.S.A.) and chilled by placing the cylinder in a freezer(- 70° C) for 15 minutes.

Partially frozen cells were ruptured at 20000 psi. The broken cell mass was collected in a 125 ml flask and 20 ml of acetone was added to it. After shaking the cells suspension thoroughly the mixture was centrifuged at 5000 rpm in Sorvall RC-5B Plus centrifuge (Dupont-Sorvall Instruments, Newark, DE, U.S.A). The supernatant was decanted into a clear flask and 20 ml fresh acetone was added to the pellet. It was then mixed and centrifuged as before. The extraction protocol is shown in Fig. 4. The acetone extracts were pooled (60 ml, approx.) and filtered through Whatman No I filter paper.

Carotenoid containing acetone solution was added to 50 mi of n-hexane and mixed in a separatory funnel. Sodium chloride was (0.5%, I 00 ml) added to maximize tbe extraction of the carotenoids. Carotenoid containing hexane solution was concentrated using an evaporator (Roto vapour-R, Brinkmann, Buchi Laborotoriums, Ontario) to 3 ml.

Yeast Cells 4 g Broken by French press

..

Add acetone, 20

l

Supernatant (s 1)

Add acetone, 20 ml ~ Pellet

t

) Centrifuge, 5 min, 5000 rpm

Vortex

cJtrifuge, 5 min, 5000 rpm

/~

Pellet Supernatant (s2) Add acetone, 20 ml

l

Vortex

Centrifuge, 5 min, 5000 rpm

Pellet

Transfer into 250 ml separatory funnel

Supernatant (s3)

~

Add sl and s2 to s3 ( 60 ml approx.) ·

Add 50 ml n-hexane + 100 ml NaCI solution (0.5%)

Aqueous phase

Concentrate, Roto- vap 3ml

/ ~

Organic phase (hexane layer)

~

Collect in a clean flask

The absorption spectrum was recorded in the region 400 to 600 nm using a spectrophotometer (Shimadzu photo spectrometer UV-260, Kyoto, Japan).

2.6.9.2 Extraction by Freeze Drying:

ln freeze-drying methods, I 0 g frozen cells were dried usmg a lyophilizer LABCONCO, Freeze Dry System, Indiana, U.S.A.). The dried powder (1 g) was treated with 6 ml of warmed dimethyl sulphoxide in a 40 ml test tube. The tube was kept in the dark at room temperature for 20 minutes by covering it with aluminum foil. The mixture was centrifuged at 5000 rpm for 5 min and the supernatant was collected. The pellet was extracted with 5 ml of additional acetone and centrifuged as before. The supernatant was collected and the pellet was treated once again with 5 ml acetone and centrifuged. The supernatants· obtained were pooled together (15 ml) were filtered through a No.1 Whatman filter paper. Petroleum ether (30 ml) and 15 ml water were added to this filtered supernatant in a separatory funnel. After thorough mixing the organic phase was allowed to separate. The bottom aqueous phase was removed and discarded. The organic phase (30 ml) containing carotenoids was dried with anhydrous sodium sulphate Na2 S04) and then concentrated using an evoporator to 3 ml as showed in Fig. 5.

2.6.9.3 Extraction by the Method of Sonication:

In sonication method fresh cells (I gm) were suspended in 2 ml acetone and sonicated for a period of 3 minutes at intervals of 30 seconds using Braun-Sonic, B Braun, Model2000 sonicator. The suspension was centrifuged for 5 minutes (5000 rpm).

Separate the supernatant (sl) from the pellet and add 2 ml of acetone to it and vortexed.

Freeze dried Cells 1 g

Centrifuge, 5 min, 5000 rpm Pellet

/~

~

Add warm DMSO, 6 ml

l

Incubated, dark, 20 mins.

Centrifuge, 5 min, 5000 rpm

Add acetone, 5 ml ..___ Pellet 2

/~

Centrifuge, 5 min, 5000 rpm

Pellet3

/~

Add 30 ml petroleum ether+ 25 ml distilled water

Aqueous phase Organic phase

Add Na2S04 (anhydrous)

l

Concentrated, roto-vap, 3 ml

This suspension was again sonicated and centrifuged as before and added the supernatant (s2) to sl. From this acetone extraction mixture 1 ml was taken to run the spectrum for the analysis of the pigment.

2.6.9.4 Extraction using Enzymes:

In enzymatic cell breakage method 1 g of wet cell mass was suspended in 2 ml of Tris HCI (pH 7) buffer or Citrate Phosphate buffer (pH 7) in a centrifuge tube (15 ml).

Lysing enzyme (3.5 mg) was added to the tube and was incubated for 24 hours in a water bath (Precision scientific Company, U.S.A.) at 25° C. Reaction mixtures were centrifuged for I 0 minutes, at a speed of 5000 rpm. The supernatant was then decanted off and 2 ml acetone was added to the pelleted cells. It was then vortexed and sonicated for 3 minutes and centrifuged again as before. The acetone layer (supernatant 1, sl) was collected in a fresh bottle, and the pellet was resuspended in 2 mJ acetone. After thorough mixing, it was once again centrifuged and the supernatant s2 was obtained, then mixed with s I in a round bottom flask and concentrated to 3 ml using an evaporator (Fig. 6).

2.6.9.4.1 Freeze and Thaw method:

In this method yeast cells were frozen at -70° C for 3 hours and were then thawed.

Thawed cells (1 g) were separately suspended in 2 ml of Tris HCI buffers with pH ranging between 7 and 9 or citrate phosphate buffer (pH, 5 to 7). These suspensions were treated with a 3.5 mg lytic enzyme (Rhyzoctonia solani) and were incubated for 24 hours at 25° C in a water bath. The incubated cell suspension was sonicated for 3 minutes and 2 ml of acetone was added to it. This suspension was centrifuged at 5000 rpm, for 10

Wet cells, lg + 2 ml buffer+ 3.5 mg enzyme - - - . Mix, incubate 24 hours, 25 deg C

1

Centrifuge, 5000 rpm, 1 0 min

Supernatant

~

1

Sonication, 3 minutes Add 2 ml acetone, Vortex

l

Supernatant S l Pellet

\

Sonication, 3 minutes Add 2 ml acetone, Vortex

1

Supernatant S2 Pellet

+

Add S 1 and S2, 4m1 Run the spectrum

minutes. The supernatant was decanted off and the pellet was suspended in 2 ml acetone.

This was vortexed and was sonicated for 3 minutes. The sonicated cell suspension was centrifuged at 5000 rpm for 10 minutes. The supernatant (sl) was separated from the pellet and 2 ml of acetone was again added to the pellet. This cell suspension was sonicated for 3 minutes and was centrifuged as mentioned above. The supernatant (s2) was mixed with sl (4 ml). Spectrum of this mixture was obtained between 200 - 600 nm. All the experiments were done in triplicates.

2.6.9.4.2 Effect of Thiol Group on Pigment Recovery:

Reducing agents such as dithiotbreitol (DTT) and beta mercapto ethanol (BME) were used at different concentrations to evaluate their effects on pigment extraction. Wet cells (1 g) were separately added to 2 ml citrate phosphate buffer (pH, 7.0) in four different tubes. The concentrations ofBME in the buffer were 50 mM, 150 mM and 200 mM. One g of cells were separately added to 2 ml citrate buffer (pH, 7.0) in four different tubes.

The concentrations of DTT in the buffer were 15, 20, 25 and 30 mM respectively. To this cell suspension 3.5 mg lysing enzyme was added and incubated for 24 hours at 25°

C. The cell suspension was sonicated for 3 minutes and then 2 mJ acetone was added to it. This was then centrifuged at a speed of 5000 rpm, for 10 minutes and decanted. Two ml of acetone was then added to the pelletted cells. The cells were vortexed and sonicated for 3 minutes and centrifuged at 5000 rpm for 10 minutes. The supernatant was separated from the pellet and 2 ml of acetone was added to the pellet. It was then vortexed, sonicated for 3 minutes and centrifuged again. The two supernatants were

mixed and the spectrum for the sample-mixture was recorded for the analysis of the pigments.

2.6.10 Measurement of Pigment:

The total carotenoid concentration in yeast cells was calculated using the formula,

. lOOAV

Carotenozd(J.Jg)l dty yeast(g)

= - - -

EW

where A is the absorbance maxima at 474 nm, Vis the total volume of the sample (ml), E is the extinction coefficient and W is the dry weight of the cells. Since the crude extracts usually contained a variety of carotenoids an average coefficient of 2100 was used in the calculations and the concentrations of the individual pigments were calculated using the method according to An et al. 1989. The absorbance values of the pigment extracts in acetone were measured by spectrophotometer. The maximum absorbance determined by scanning from 600 to 300 run in a Shimadzu Ultra Violet 260 Recording spectrophotometer. Identification of the individual pigments was done by comparison of their absorption maxima with those of standard carotenoids reported by other researchers (Davies, 1976; Bauerfeind and Klaui, 1981).

2.6.1 1 Thin Layer Chromatography:

The pigments were separated by means of Thin Layer Chromatograph. Pre-coated silica gel (Whatman International Ltd., Maidstone, England) plates were used to chromatograph the samples. The solvent used was a 10% toluene mixed in 90%

petroleum ether (v/v). The spotted TLC plate was developed in this solvent until the solvent front was about 1 em below the top of the plate. The spots were visuaHzed under

ultra-violet rays and also by iodine vapours. Rr (Retardation factor) values were calculated by using the ratio of the distance traveled by the substance to the distance traveled by the soJvent.

CHAPTER3

RESULTS AND DISCUSSION 3.1 Growth and pigment production by R.rubra mutants:

The growth rate was determined in YM broth. R. rubra mutants (Ml, M2 and M3), showed Jess growth than the parent TPI. The generation times for mutants M1 and M2 were 12.0 and 11.52 hours, respectively. Figure 7 illustrates the generation times for the wild type and mutant yeasts. The time required for the population to double in the case of TP 1 was less, indicating faster growth rate compared to the mutants. R. rubra TPI had a shorter generation time and greater biomass yield than that of P. rhodozyma,

in an earlier investigation (Sangha, 1994).

The growth curves for the mutants and the wild type organisms at 15° C are shown in Fig.8. All the isolates showed a Jag period of about fifteen hours as shown in the figure. Figure 9 examines the growth curves at 25° C. The cell yields were greater for cultures grown at 25° C (Table 1 ). The time to reach stationary phase for the mutants Ml, M2 and M3 were 42.8, 41.2 and 43.2 hours, respectively (Fig. 10). M3 showed more time as parent TPI to reach stationary phase than the other two mutants, Ml and M2. This figure shows the differences in times to reach stationary phase by the mutants and the wild type yeasts.

3.1.1 Growth on Cane Molasses and Peat Extracts:

Greater biomass yield was obtained upon addition of peat extract to the YM broth.

Figure 11 shows the effect of 1 % peat extract, on M 1, M2, M3 and TP 1. The yields

15

14.5 14 .2

14 13.5

fA ~

13

~

012.5

.c::

c

12

~

11.5 i= 11

10.5 10 9.5 9

TP1 Rm Rt M1 M2 M3 Yeast sample

Fig. 7. Generation times of different yeasts.

Yeast cells were growing in liquid media as described under Materials and Methods.

Each of tbe data points represents the mean value of three determinations. The standard errors in the mean for TPl, Rm, Rt, Ml, M2 and M3 are± 0.12, ± 0.23, ± 0.20, ± 0.31, ± 0.25 and ± 0.11 hour, respectively.

2.5

2.0

• _....

_• ^• •

E _{c 1.5 ~ M3} M2

• • ^• •

0 - - ODvsTime

•

0 A

<D

•

-

~ _(/) c 1.0

• •

Q) A

0 ^A

u (1J 0.5 ...

· c.

0

0.0

0 20 40 60

Time (Hours)

Fig.8 Growth of yeasts on YM-broth at 15° C.

Erlenmayer flasks (500 ml) containing 300 ml of the Liquid medium were inoculated with freshly grown yeasts on RB agar and incubated on a Psychrotherm, agitated at150 rpm.

The solid curve is shown for Ml only. The data points are averages of three determinations (standard deviation,± 0.5 O.D. units).

2.5

•

2.0

•

•

E _{0 M1}

c:

0 1.5

...

0

•

<0

-

~ <n 1.0

c: Q)

0

ro

~ a.

0 0.0

0 20 40 60

Time (Hours)

Fig. 9 Growth of different yeasts on YM-broth at 25° C.

Growth conditions were similar to those given in the figure caption for Fig. 8 except temperature. The figure represents growth measured at different time intervals. The solid curve is shown for TPI, yeast only. The data points are averages of 3 determinations (standard deviation,± 0.5 O.D. units).

49.5

48.5 47.5

(/)

'- 46.5

::l 0 45.5 .c

c:::

Q) 44.5

E

t- 43.5 42.5 41.5 40.5

TP1

Rm Rt M1

M3

Fig. 10 Time needed for achieving stationary phase by different yeasts in liquid cultures.

Each of the data points represents the mean value of three determinations. The standard errors in the mean for TPl, Rm, Rt, Ml, M2 and M3 are± 0.2, ± 0.3, ± 0.3, ± 0.5, ± 0.1 and± 0.1, respectively.

Table I. Biomass yield (giL) in wild type and mutant yeasts at various temperatures.

Yeast TPl Rm Rt Ml M2 M3

Temp. Biomass yeild

15 4.0± 0.2 4.1 ± 0.3 3.5 ±0.2 4.6± 0.2 3.7±0.1 3.2 ± 0.3 20 8.3 ±0.2 6.9 ± 0.1 8.0 ± 0.1 7.6 ± 0.1 8.4 ± 0.2 7.3 ± 0.2 25 10.2±0.1 11.2 ± 0.6 12.5 ± 0.4 9.3 ± 0.3 8.6±0.2 8.4 ± 0.2 28 12.5 ± 0.2 12.0 ± 0.4 13.1±0.3 11.0 ± 0.3 10.2 ± 0.2 9.5±0.1 30 9.4 ± 0.2 10.9:!: 0.1 12.7 ± 0.2 7.1:!:0.1 7.1 ± 0.2 7.6± 0.2 35 7.0± 0.1 10.3 ± 0.3 11.2 ± 0.3 6.3 ± 0.3 5.9±0.2 6.1 ± 0.2

Yeasts were grown in a liquid media in flasks (500 ml) containing 300 mJ YM-broth.

Inoculated flasks were incubated at various temperatures (15 to 35° C) separately, in a Psychrotherm shaker at 150 rpm. Cells were collected by centrifugation after 5 days.

Wet weights and dry weights were determined in pre-weighed glass centrifuge tubes.

Each experimental point represents average of three determinations. Errors given are standard deviations.

-

13 ¹²

-

¹¹

-

-

>. 9

fll

fll

8

cu

E

m

₆

5

TP1

Fig. 11 Biomass yield of different yeasts grown in YM-broth with 1 % peat extract.

Yeast cells were grown in liquid cultures in Erlenmeyer flasks (500 ml) containing 300 ml liquid media inoculated with different yeasts. Culture flasks were incubated at 25° C and shaken in a Psycbrothenn at 150 rpm. Yeast cells were harvested by centrifugation as described under Materials and Methods.

Each experimental point represents an average of three readings. The standard error in the mean for TPl, Rm, Rt, Ml, M2 and M3 are± 0.25, 0.32, 0.42, 0.51, 0.23 and 0.39 giL, dry weight, respectively.

were 7.87, 8.29, 11.42 and 9.54 giL (dry weight), respectively. The effect of 2 % peat extract in YM broth was observed to be better for larger biomass yields. At this concentration M2 and M3 resulted in biomass yield of 9.71 and 9.84 giL, respectively.

In contrast under similar conditions the parent TPl gave 10.12 giL, as shown in Fig. 12. Further increases in peat extract concentrations for the growth medium did not give corresponding increases in the biomass yields except in the case of M I (Fig. 13).

The yeasts were able to utilize a wide variety of inorganic nitrogen sources with an optimum growth in the presence of ammonium sulphate and ammonium hydroxide (Sangha, 1994). However, an organic nitrogen source like peptone was assimilated much better than an inorganic-nitrogen source (Sangha, 1994). Abour-Zeid and Yousef (1972) also reported similar behavior with Streptomyces caespitosus. The yeast preferred molasses may be because of the presence of lower amount of reducing sugars in the peat hydrolysate. Anderson (I 979) grew Candida uti/is on a commercial scale using sulfite waste liquor. Nitrogen supplementation of sulphite waste liquor in the form of urea or ammonium sulfate and phosphorus as phosphoric acid was found to enhance the biomass yield and substrate consumption (Simard and Cameron, 1974).

Figure 14 shows the effect of different concentrations of cane molasses in YM broth on biomass yield. With 1 and 2 % cane molasses the mutants and the parent organism showed much greater cell growth than with the higher concentration (3 %).

The biomass yield by mutants showed considerable variation as shown in figure 14.

Earlier workers found that cane molasses were better than beet molasses in supporting the growth of the yeast (Peppler, 1979). It is postulated that the higher

Fig. 12 Biomass yield of yeast cells grown in YM-broth with 2% peat extract.

Growth conditions were similar to those described for caption for Fig. II except the concentration of peat extract in the growth medium.

Each experimental data represents an average of three readings. The standard error of mean for TPI, Rm, Rt, Ml, M2 and M3 are ± 0.39, 0.23, 0.51, 0.42, 0.23 and 0.32 giL, dry weight, respectively.

9.5 ---

-

⁹

-

^8.5

-

_{- 8}

·-

_7.5

en en ₇ E

6.5

·-

m ₆

5.5

TP1 Rm Rt M1 M2 M3 Yeast sample

Fig. 13 Effect of3% peat extract on the biomass yield of yeast cells.

Growth conditions were similar to those described for caption for Fig. 11 except the concentration of peat extract in the growth medium.

Each experimental data represents an average of three readings. The standard error of mean for TPl, Rm, Rt, Ml, M2 and M3 are ± 0.23, 0.25, 0.32, 0.39, 0.42 and 0.32 giL, dry weight, respectively.